Determination of the optimal number of control genes for normalization. (a) Pairwise variation (Vn/n + 1) analysis between the normalization factors NF
and NFn + 1 to determine the number of control genes required for accurate normalization (arrowhead = optimal number of control genes for normalization). (b) Selected scatterplots of normalization factors before (x-axis) and after (y-axis) inclusion of an (n + 1)th control gene (r = Spearman rank correlation coefficient). Low variation values, V, correspond to high correlation coefficients. It is clear that there is no need to include more than three, four or five control genes for fibroblast (A), neuroblastoma (B) and the normal pooled tissues (D), respectively. In contrast, panel C demonstrates that inclusion of at least a fourth control gene is required for the normal pooled tissues.